Chimeric antigen receptors targeting B-cell maturation antigen

ABSTRACT

The invention provides an isolated and purified nucleic acid sequence encoding a chimeric antigen receptor (CAR) directed against B-cell Maturation Antigen (BCMA). The invention also provides host cells, such as T-cells or natural killer (NK) cells, expressing the CAR and methods for destroying multiple myeloma cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.15/692,473, filed Aug. 31, 2017, now U.S. Pat. No. 10,767,184 which is acontinuation of U.S. application Ser. No. 14/389,677, filed Sep. 30,2014, now U.S. Pat. No. 9,765,342, which is the U.S. National Phase ofInternational Patent Application No. PCT/US2013/032029, filed Mar. 15,2013, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/622,600, filed Apr. 11, 2012, each of which is incorporated byreference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numberZIABC011417 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 42,722 Byte ASCII (Text) file named“745739_ST25.TXT,” created on Oct. 18, 2019.

BACKGROUND OF THE INVENTION

Multiple myeloma (MM) is a malignancy characterized by an accumulationof clonal plasma cells (see, e.g., Palumbo et al., New England J. Med.,364(11): 1046-1060 (2011), and Lonial et al., Clinical Cancer Res.,17(6): 1264-1277 (2011)). Current therapies for MM often causeremissions, but nearly all patients eventually relapse and die (see,e.g., Lonial et al., supra, and Rajkumar, Nature Rev. Clinical Oncol.,8(8): 479-491 (2011)). Allogeneic hematopoietic stem celltransplantation has been shown to induce immune-mediated elimination ofmyeloma cells; however, the toxicity of this approach is high, and fewpatients are cured (see, e.g., Lonial et al., supra, and Salit et al.,Clin. Lymphoma, Myeloma, and Leukemia, 11(3): 247-252 (2011)).Currently, there are no clinically effective, FDA-approved monoclonalantibody or autologous T-cell therapies for MM (see, e.g., Richardson etal., British J. Haematology, 154(6): 745-754 (2011), and Yi, CancerJournal, 15(6): 502-510 (2009)).

Adoptive transfer of T-cells genetically modified to recognizemalignancy-associated antigens is showing promise as a new approach totreating cancer (see, e.g., Morgan et al., Science, 314(5796): 126-129(2006); Brenner et al., Current Opinion in Immunology, 22(2): 251-257(2010); Rosenberg et al., Nature Reviews Cancer, 8(4): 299-308 (2008),Kershaw et al., Nature Reviews Immunology, 5(12): 928-940 (2005); andPule et al., Nature Medicine, 14(11): 1264-1270 (2008)). T-cells can begenetically modified to express chimeric antigen receptors (CARs), whichare fusion proteins comprised of an antigen recognition moiety andT-cell activation domains (see, e.g., Kershaw et al., supra, Eshhar etal., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993), and Sadelain etal., Curr. Opin. Immunol., 21(2): 215-223 (2009)).

For B-cell lineage malignancies, extensive progress has been made indeveloping adoptive T-cell approaches that utilize anti-CD19 CARs (see,e.g., Jensen et al., Biology of Blood and Marrow Transplantation, 16:1245-1256 (2010); Kochenderfer et al., Blood, 116(20): 4099-4102 (2010);Porter et al., The New England Journal of Medicine, 365(8): 725-733(2011); Savoldo et al., Journal of Clinical Investigation, 121(5):1822-1826 (2011), Cooper et al., Blood, 101(4): 1637-1644 (2003);Brentjens et al., Nature Medicine, 9(3): 279-286 (2003); and Kalos etal., Science Translational Medicine, 3(95): 95ra73 (2011)). Adoptivelytransferred anti-CD19-CAR-transduced T-cells have cured leukemia andlymphoma in mice (see, e.g., Cheadle et al., Journal of Immunology,184(4): 1885-1896 (2010); Brentjens et al., Clinical Cancer Research,13(18 Pt 1): 5426-5435 (2007); and Kochenderfer et al., Blood, 116(19):3875-3886 (2010)). In early clinical trials, adoptively transferredT-cells transduced with anti-CD19 CARs eradicated normal and malignantB-cells in patients with leukemia and lymphoma (see, e.g., Kochenderferet 1, Blood, 116(20): 4099-4102 (2010); Porter et al., supra, Brentjenset al., Blood, 118(18): 4817-4828 (2011); and Kochenderfer et al.,Blood, Dec. 8, 2011 (epublication ahead of print (2012)). CD19, however,is only rarely expressed on the malignant plasma cells of multiplemyeloma (see, e.g., Gupta et al., Amer. J. Clin. Pathology, 132(5):728-732 (2009); and Lin et al., Amer. J. Clin. Pathology, 121(4):482-488 (2004)).

Thus, there remains a need for compositions that can be used in methodsto treat multiple myeloma. This invention provides such compositions andmethods.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated or purified nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises anantigen recognition moiety and a T-cell activation moiety, and whereinthe antigen recognition moiety is directed against B-cell MaturationAntigen (BCMA).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A and 1B are graphs which depict experimental data illustratingthe expression pattern of BCMA across a variety of human cell types, asdetermined using quantitative PCR. The results are expressed as thenumber of BCMA cDNA copies per 10⁵ actin cDNA copies.

FIGS. 2A-2L are graphs which depict experimental data illustrating thatcell-surface BCMA expression was detected on multiple myeloma celllines, but not on other types of cells, as described in Example 1. Forall plots, the solid line represents staining with anti-BCMA antibodies,and the dashed line represents staining with isotype-matched controlantibodies. All plots were gated on live cells.

FIG. 3A is a diagram which depicts a nucleic acid construct encoding ananti-BCMA CAR. From the N-terminus to the C-terminus, the anti-BCMA CARincludes an anti-BCMA scFv, the hinge and transmembrane regions of theCD8α molecule, the cytoplasmic portion of the CD28 molecule, and thecytoplasmic portion of the CD3ζ molecule.

FIGS. 3B-3D are graphs which depict experimental data illustrating thatthe anti-bcma1 CAR, the anti-bcma2 CAR, and the SP6 CAR (described inExample 2) are expressed on the surface of T-cells. Minimal anti-Fabstaining occurred on untransduced (UT) cells. The plots are gated onCD3⁺ lymphocytes. The numbers on the plots are the percentages of cellsin each quadrant.

FIGS. 4A-4C are graphs which depict experimental data illustrating thatT-cells expressing anti-BCMA CARs degranulate T-cells in a BCMA-specificmanner, as described Example 3. The plots are gated on live CD3+lymphocytes. The numbers on the plots are the percentages of cells ineach quadrant.

FIGS. 5A-5D are graphs which depict experimental data illustrating thatT-cells expressing anti-BCMA CARs degranulate T-cells in a BCMA-specificmanner, as described Example 3. The plots are gated on live CD3+lymphocytes. The numbers on the plots are the percentages of cells ineach quadrant.

FIGS. 6A-6C are graphs which depict experimental data illustrating thatT-cells expressing anti-BCMA CARs produce the cytokines IFNγ, IL-2, andTNF in a BCMA-specific manner, as described Example 3. The plots aregated on live CD3+ lymphocytes. The numbers on the plots are thepercentages of cells in each quadrant.

FIG. 7A is a graph which depicts experimental data illustrating thatT-cells expressing the anti-bcma2 CAR proliferated specifically inresponse to BCMA. FIG. 7B is a graph which depicts experimental dataillustrating that T-cells expressing the SP6 CAR did not proliferatespecifically in response to BCMA.

FIGS. 7C and 7D are graphs which depict experimental data illustratingthat T-cells from Donor A expressing the anti-bcma2 CAR specificallykilled the multiple myeloma cell lines H929 (FIG. 6C) and RPMI8226 (FIG.6D) in a four-hour cytotoxicity assay at various effector:target cellratios. T-cells transduced with the negative control SP6 CAR inducedmuch lower levels of cytotoxicity at all effector:target ratios. For alleffector:target ratios, the cytotoxicity was determined in duplicate,and the results are displayed as the mean+/− the standard error of themean.

FIG. 8A is a graph which depicts experimental data illustrating thatBCMA is expressed on the surface of primary bone marrow multiple myelomacells from Myeloma Patient 3, as described in Example 5. The plot isgated on CD38^(high) CD56⁺ plasma cells, which made up 40% of the bonemarrow cells.

FIG. 8B is a graph which depicts experimental data illustrating thatallogeneic T-cells transduced with the anti-bcma2 CAR from Donor Cproduced IFNγ after co-culture with the unmanipulated bone marrow cellsof Myeloma Patient 3, as described in Example 5. FIG. 7B alsoillustrates that T-cells from the same allogeneic donor expressing theanti-bcma2 CAR produced much less IFNγ when they were cultured withperipheral blood mononuclear cell (PBMC) from Myeloma Patient 3. Inaddition, T-cells from Donor C expressing the SP6 CAR did notspecifically recognize the bone marrow of Myeloma Patient 3.

FIG. 8C is a graph which depicts experimental data illustrating that aplasmacytoma resected from Myeloma Patient 1 consisted of 93% plasmacells, and these primary plasma cells expressed BCMA, as revealed byflow cytometry for BCMA (solid line) and isotype-matched controlstaining (dashed line). The plot is gated on plasma cells.

FIG. 8D is a graph which depicts experimental data illustrating thatT-cells from Myeloma Patient 1 expressing the anti-bcma2 CAR producedIFNγ specifically in response to autologous plasmacytoma cells.

FIG. 8E is a graph which depicts experimental data illustrating thatT-cells from Myeloma Patient 1 expressing the anti-bcma2 CARspecifically killed autologous plasmacytoma cells at low effector totarget ratios. In contrast, T-cells from Myeloma Patient 1 expressingthe SP6 CAR exhibited low levels of cytotoxicity against autologousplasmacytoma cells. For all effector:target ratios, the cytotoxicity wasdetermined in duplicate, and the results are displayed as the mean+/−the standard error of the mean.

FIG. 9A is a graph which depicts experimental data illustrating thatT-cells transduced with the anti-bcma2 CAR can destroy establishedmultiple myeloma tumors in mice. FIG. 9B is a graph which depicts thesurvival of tumor-bearing mice treated with T-cells expressing theanti-bcma2 CAR as compared to controls.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated or purified nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises anantigen recognition moiety and a T-cell activation moiety. A chimericantigen receptor (CAR) is an artificially constructed hybrid protein orpolypeptide containing an antigen binding domain of an antibody (e.g., asingle chain variable fragment (scFv)) linked to T-cell signaling orT-cell activation domains. CARs have the ability to redirect T-cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT-cells expressing CARs the ability to recognize an antigen independentof antigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARs advantageously do not dimerizewith endogenous T-cell receptor (TCR) alpha and beta chains.

“Nucleic acid sequence” is intended to encompass a polymer of DNA orRNA, i.e., a polynucleotide, which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “nucleic acid” and “polynucleotide” as usedherein refer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecule, and thus include double- andsingle-stranded DNA, and double- and single-stranded RNA. The termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to methylated and/or capped polynucleotides.

By “isolated” is meant the removal of a nucleic acid from its naturalenvironment. By “purified” is meant that a given nucleic acid, whetherone that has been removed from nature (including genomic DNA and mRNA)or synthesized (including cDNA) and/or amplified under laboratoryconditions, has been increased in purity, wherein “purity” is a relativeterm, not “absolute purity.” It is to be understood, however, thatnucleic acids and proteins may be formulated with diluents or adjuvantsand still for practical purposes be isolated. For example, nucleic acidstypically are mixed with an acceptable carrier or diluent when used forintroduction into cells.

The inventive nucleic acid sequence encodes a CAR which comprises anantigen recognition moiety that is directed against B-cell MaturationAntigen (BCMA, also known as CD269). BCMA is a member of the tumornecrosis factor receptor superfamily (see, e.g., Thompson et al., J.Exp. Medicine, 192(1): 129-135 (2000), and Mackay et al., Annu. Rev.Immunol., 21: 231-264 (2003)). BCMA binds B-cell activating factor(BAFF) and a proliferation inducing ligand (APRIL) (see, e.g., Mackay etal., supra, and Kalled et al., Immunological Reviews, 204: 43-54(2005)). Among nonmalignant cells, BCMA has been reported to beexpressed mostly in plasma cells and subsets of mature B-cells (see,e.g., Laabi et al., EMBO J., 11(11): 3897-3904 (1992); Laabi et al.,Nucleic Acids Res., 22(7): 1147-1154 (1994); Kalled et al., supra;O'Connor et al., J. Exp. Medicine, 199(1): 91-97 (2004); and Ng et al.,J. Immunol., 173(2): 807-817 (2004)). Mice deficient in BCMA are healthyand have normal numbers of B-cells, but the survival of long-livedplasma cells is impaired (see, e.g., O'Connor et al, supra; Xu et al.,Mol. Cell. Biol., 21(12): 4067-4074 (2001); and Schiemann et al.,Science, 293(5537): 2111-2114 (2001)). BCMA RNA has been detecteduniversally in multiple myeloma cells, and BCMA protein has beendetected on the surface of plasma cells from multiple myeloma patientsby several investigators (see, e.g., Novak et al., Blood, 103(2):689-694 (2004); Neri et al., Clinical Cancer Research, 13(19): 5903-5909(2007); Bellucci et al., Blood, 105(10): 3945-3950 (2005); and Moreauxet al., Blood, 103(8): 3148-3157 (2004)).

The inventive nucleic acid sequence encodes a CAR which comprises anantigen recognition moiety that contains a monoclonal antibody directedagainst BCMA, or an antigen-binding portion thereof. The term“monoclonal antibodies,” as used herein, refers to antibodies that areproduced by a single clone of B-cells and bind to the same epitope. Incontrast, “polyclonal antibodies” refer to a population of antibodiesthat are produced by different B-cells and bind to different epitopes ofthe same antigen. The antigen recognition moiety of the CAR encoded bythe inventive nucleic acid sequence can be a whole antibody or anantibody fragment. A whole antibody typically consists of fourpolypeptides: two identical copies of a heavy (H) chain polypeptide andtwo identical copies of a light (L) chain polypeptide. Each of the heavychains contains one N-terminal variable (VH) region and three C-terminalconstant (CH1, CH2 and CH3) regions, and each light chain contains oneN-terminal variable (VL) region and one C-terminal constant (CL) region.The variable regions of each pair of light and heavy chains form theantigen binding site of an antibody. The VH and VL regions have the samegeneral structure, with each region comprising four framework regions,whose sequences are relatively conserved. The framework regions areconnected by three complementarity determining regions (CDRs). The threeCDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” ofan antibody, which is responsible for antigen binding.

The terms “fragment of an antibody,” “antibody fragment,” “functionalfragment of an antibody,” and “antigen-binding portion” are usedinterchangeably herein to mean one or more fragments or portions of anantibody that retain the ability to specifically bind to an antigen(see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129(2005)). The antigen recognition moiety of the CAR encoded by theinventive nucleic acid sequence can contain any BCMA-binding antibodyfragment. The antibody fragment desirably comprises, for example, one ormore CDRs, the variable region (or portions thereof), the constantregion (or portions thereof), or combinations thereof. Examples ofantibody fragments include, but are not limited to, (i) a Fab fragment,which is a monovalent fragment consisting of the VL, VH, CL, and CH1domains; (ii) a F(ab′)2 fragment, which is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody; (iv) a single chain Fv (scFv), which is amonovalent molecule consisting of the two domains of the Fv fragment(i.e., VL and VH) joined by a synthetic linker which enables the twodomains to be synthesized as a single polypeptide chain (see, e.g., Birdet al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad.Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol.,16: 778 (1998)) and (v) a diabody, which is a dimer of polypeptidechains, wherein each polypeptide chain comprises a VH connected to a VLby a peptide linker that is too short to allow pairing between the VHand VL on the same polypeptide chain, thereby driving the pairingbetween the complementary domains on different VH-VL polypeptide chainsto generate a dimeric molecule having two functional antigen bindingsites. Antibody fragments are known in the art and are described in moredetail in, e.g., U.S. Patent Application Publication 2009/0093024 A1. Ina preferred embodiment, the antigen recognition moiety of the CARencoded by the inventive nucleic acid sequence comprises an anti-BCMAsingle chain Fv (scFv).

An antigen-binding portion or fragment of a monoclonal antibody can beof any size so long as the portion binds to BCMA. In this respect, anantigen binding portion or fragment of the monoclonal antibody directedagainst BCMA (also referred to herein as an “anti-BCMA monoclonalantibody”) desirably comprises between about 5 and 18 amino acids (e.g.,about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a rangedefined by any two of the foregoing values).

In one embodiment, the inventive nucleic acid sequence encodes anantigen recognition moiety that comprises a variable region of ananti-BCMA monoclonal antibody. In this respect, the antigen recognitionmoiety comprises a light chain variable region, a heavy chain variableregion, or both a light chain variable region and a heavy chain variableregion of an anti-BCMA monoclonal antibody. Preferably, the antigenrecognition moiety of the CAR encoded by the inventive nucleic acidsequence comprises a light chain variable region and a heavy chainvariable region of an anti-BCMA monoclonal antibody. Heavy and lightchain monoclonal antibody amino acid sequences that bind to BCMA aredisclosed in, e.g., International Patent Application Publication WO2010/104949.

In another embodiment, the inventive nucleic acid sequence encodes a CARwhich comprises a signal sequence. The signal sequence may be positionedat the amino terminus of the antigen recognition moiety (e.g., thevariable region of the anti-BCMA antibody). The signal sequence maycomprise any suitable signal sequence. In one embodiment, the signalsequence is a human granulocyte-macrophage colony-stimulating factor(GM-CSF) receptor sequence or a CD8α signal sequence.

In another embodiment, the CAR comprises a hinge sequence. One ofordinary skill in the art will appreciate that a hinge sequence is ashort sequence of amino acids that facilitates antibody flexibility(see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). Thehinge sequence may be positioned between the antigen recognition moiety(e.g., an anti-BCMA scFv) and the T-cell activation moiety. The hingesequence can be any suitable sequence derived or obtained from anysuitable molecule. In one embodiment, for example, the hinge sequence isderived from the human CD8α molecule or a CD28 molecule.

The inventive nucleic acid sequence encodes a CAR comprising a T-cellactivation moiety. The T-cell activation moiety can be any suitablemoiety derived or obtained from any suitable molecule. In oneembodiment, for example, the T-cell activation moiety comprises atransmembrane domain. The transmembrane domain can be any transmembranedomain derived or obtained from any molecule known in the art. Forexample, the transmembrane domain can be obtained or derived from a CD8αmolecule or a CD28 molecule. CD8 is a transmembrane glycoprotein thatserves as a co-receptor for the T-cell receptor (TCR), and is expressedprimarily on the surface of cytotoxic T-cells. The most common form ofCD8 exists as a dimer composed of a CD8α and CD8β chain. CD28 isexpressed on T-cells and provides co-stimulatory signals required forT-cell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2).In a preferred embodiment, the CD8α and CD28 are human.

In addition to the transmembrane domain, the T-cell activation moietyfurther comprises an intracellular (i.e., cytoplasmic) T-cell signalingdomain. The intercellular T-cell signaling domain can be obtained orderived from a CD28 molecule, a CD3 zeta (ζ) molecule or modifiedversions thereof, a human Fc receptor gamma (FcRγ) chain, a CD27molecule, an OX40 molecule, a 4-1BB molecule, or other intracellularsignaling molecules known in the art. As discussed above, CD28 is aT-cell marker important in T-cell co-stimulation. CD3ζ associates withTCRs to produce a signal and contains immunoreceptor tyrosine-basedactivation motifs (ITAMs). 4-1BB, also known as CD137, transmits apotent costimulatory signal to T-cells, promoting differentiation andenhancing long-term survival of T lymphocytes. In a preferredembodiment, the CD28, CD3 zeta, 4-1BB, OX40, and CD27 are human.

The T-cell activation domain of the CAR encoded by the inventive nucleicacid sequence can comprise any one of aforementioned transmembranedomains and any one or more of the aforementioned intercellular T-cellsignaling domains in any combination. For example, the inventive nucleicacid sequence can encode a CAR comprising a CD28 transmembrane domainand intracellular T-cell signaling domains of CD28 and CD3 zeta.Alternatively, for example, the inventive nucleic acid sequence canencode a CAR comprising a CD8α transmembrane domain and intracellularT-cell signaling domains of CD28, CD3 zeta, the Fc receptor gamma (FcRγ)chain, and/or 4-1BB.

In one embodiment, the inventive nucleic acid sequence encodes a CARwhich comprises, from 5′ to 3′, a granulocyte-macrophage colonystimulating factor receptor (GM-CSF receptor) signal sequence, ananti-BCMA scFv, the hinge and transmembrane regions of the human CD8αmolecule, the cytoplasmic T-cell signaling domain of the human CD28molecule, and T-cell signaling domain of the human CD3ζ molecule. Inanother embodiment, the inventive nucleic acid sequence encodes a CARwhich comprises, from 5′ to 3′, a human CD8α signal sequence, ananti-BCMA scFv, the hinge and transmembrane regions of the human CD8αmolecule, the cytoplasmic T-cell signaling domain of the human CD28molecule, and T-cell signaling domain of the human CD3ζ molecule. Inanother embodiment, the inventive nucleic acid sequence encodes a CARwhich comprises, from 5′ to 3′, a human CD8α signal sequence, ananti-BCMA scFv, the hinge and transmembrane regions of the human CD8αmolecule, the cytoplasmic T-cell signaling domain of the human 4-1BBmolecule and/or the cytoplasmic T-cell signaling domain of the humanOX40 molecule, and T-cell signaling domain of the human CD3ζ molecule.For example, the inventive nucleic acid sequence comprises or consistsof the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ IDNO: 3.

The invention further provides an isolated or purified chimeric antigenreceptor (CAR) encoded by the inventive nucleic acid sequence.

The nucleic acid sequence of the invention can encode a CAR of anylength, i.e., the CAR can comprise any number of amino acids, providedthat the CAR retains its biological activity, e.g., the ability tospecifically bind to antigen, detect diseased cells in a mammal, ortreat or prevent disease in a mammal, etc. For example, the CAR cancomprise 50 or more (e.g., 60 or more, 100 or more, or 500 or more)amino acids, but less than 1,000 (e.g., 900 or less, 800 or less, 700 orless, or 600 or less) amino acids. Preferably, the CAR is about 50 toabout 700 amino acids (e.g., about 70, about 80, about 90, about 150,about 200, about 300, about 400, about 550, or about 650 amino acids),about 100 to about 500 amino acids (e.g., about 125, about 175, about225, about 250, about 275, about 325, about 350, about 375, about 425,about 450, or about 475 amino acids), or a range defined by any two ofthe foregoing values.

Included in the scope of the invention are nucleic acid sequences thatencode functional portions of the CAR described herein. The term“functional portion,” when used in reference to a CAR, refers to anypart or fragment of the CAR of the invention, which part or fragmentretains the biological activity of the CAR of which it is a part (theparent CAR). Functional portions encompass, for example, those parts ofa CAR that retain the ability to recognize target cells, or detect,treat, or prevent a disease, to a similar extent, the same extent, or toa higher extent, as the parent CAR. In reference to a nucleic acidsequence encoding the parent CAR, a nucleic acid sequence encoding afunctional portion of the CAR can encode a protein comprising, forexample, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of theparent CAR.

The inventive nucleic acid sequence can encode a functional portion of aCAR that contains additional amino acids at the amino or carboxyterminus of the portion, or at both termini, which additional aminoacids are not found in the amino acid sequence of the parent CAR.Desirably, the additional amino acids do not interfere with thebiological function of the functional portion, e.g., recognize targetcells, detect cancer, treat or prevent cancer, etc. More desirably, theadditional amino acids enhance the biological activity of the CAR, ascompared to the biological activity of the parent CAR.

The invention also provides nucleic acid sequences encoding functionalvariants of the aforementioned CAR. The term “functional variant,” asused herein, refers to a CAR, a polypeptide, or a protein havingsubstantial or significant sequence identity or similarity to the CARencoded by the inventive nucleic acid sequence, which functional variantretains the biological activity of the CAR of which it is a variant.Functional variants encompass, for example, those variants of the CARdescribed herein (the parent CAR) that retain the ability to recognizetarget cells to a similar extent, the same extent, or to a higherextent, as the parent CAR. In reference to a nucleic acid sequenceencoding the parent CAR, a nucleic acid sequence encoding a functionalvariant of the CAR can be for example, about 10% identical, about 25%identical, about 30% identical, about 50% identical, about 65%identical, about 80% identical, about 90% identical, about 95%identical, or about 99% identical to the nucleic acid sequence encodingthe parent CAR.

A functional variant can, for example, comprise the amino acid sequenceof the CAR encoded by the inventive nucleic acid sequence with at leastone conservative amino acid substitution. The phrase “conservative aminoacid substitution” or “conservative mutation” refers to the replacementof one amino acid by another amino acid with a common property. Afunctional way to define common properties between individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz, G. E.and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag,New York (1979)). According to such analyses, groups of amino acids maybe defined where amino acids within a group exchange preferentially witheach other, and therefore resemble each other most in their impact onthe overall protein structure (Schulz, G. E. and Schirmer, R. H.,supra). Examples of conservative mutations include amino acidsubstitutions of amino acids within the sub-groups above, for example,lysine for arginine and vice versa such that a positive charge may bemaintained; glutamic acid for aspartic acid and vice versa such that anegative charge may be maintained; serine for threonine such that a free—OH can be maintained; and glutamine for asparagine such that a free—NH₂ can be maintained.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent CAR with at least one non-conservativeamino acid substitution. “Non-conservative mutations” involve amino acidsubstitutions between different groups, for example, lysine fortryptophan, or phenylalanine for serine, etc. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with, or inhibit the biological activity of, the functionalvariant. The non-conservative amino acid substitution may enhance thebiological activity of the functional variant, such that the biologicalactivity of the functional variant is increased as compared to theparent CAR.

The inventive nucleic acid sequence can encode a CAR (includingfunctional portions and functional variants thereof) that comprisessynthetic amino acids in place of one or more naturally-occurring aminoacids. Such synthetic amino acids are known in the art, and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The inventive nucleic acid sequence can encode a CAR (includingfunctional portions and functional variants thereof) which isglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into anacid addition salt and/or optionally dimerized or polymerized, orconjugated.

In a preferred embodiment, the inventive nucleic acid sequence encodes aCAR that comprises or consists of the amino acid sequence of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID NO: 12.

The inventive nucleic acid sequence can be generated using methods knownin the art. For example, nucleic acid sequences, polypeptides, andproteins can be recombinantly produced using standard recombinant DNAmethodology (see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.2001; and Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994). Further, asynthetically produced nucleic acid sequence encoding the CAR can beisolated and/or purified from a source, such as a plant, a bacterium, aninsect, or a mammal, e.g., a rat, a human, etc. Methods of isolation andpurification are well-known in the art. Alternatively, the nucleic acidsequences described herein can be commercially synthesized. In thisrespect, the inventive nucleic acid sequence can be synthetic,recombinant, isolated, and/or purified.

The invention also provides a vector comprising the nucleic acidsequence encoding the inventive CAR. The vector can be, for example, aplasmid, a cosmid, a viral vector (e.g., retroviral or adenoviral), or aphage. Suitable vectors and methods of vector preparation are well knownin the art (see, e.g., Sambrook et al., supra, and Ausubel et al.,supra).

In addition to the inventive nucleic acid sequence encoding the CAR, thevector preferably comprises expression control sequences, such aspromoters, enhancers, polyadenylation signals, transcriptionterminators, internal ribosome entry sites (IRES), and the like, thatprovide for the expression of the nucleic acid sequence in a host cell.Exemplary expression control sequences are known in the art anddescribed in, for example, Goeddel, Gene Expression Technology: Methodsin Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

A large number of promoters, including constitutive, inducible, andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources of promoters include forexample, virus, mammal, insect, plant, yeast, and bacteria, and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available, for example, fromdepositories such as the ATCC as well as other commercial or individualsources. Promoters can be unidirectional (i.e., initiate transcriptionin one direction) or bi-directional (i.e., initiate transcription ineither a 3′ or 5′ direction). Non-limiting examples of promotersinclude, for example, the T7 bacterial expression system, pBAD (araA)bacterial expression system, the cytomegalovirus (CMV) promoter, theSV40 promoter, and the RSV promoter. Inducible promoters include, forexample, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), theEcdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.),LACSWITCH™ System (Stratagene, San Diego, Calif.), and the Cre-ERTtamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res.,27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No.7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308: 123-144(2005)).

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a nucleic acid sequence towhich it is operably linked. Enhancers can be located many kilobasesaway from the coding region of the nucleic acid sequence and can mediatethe binding of regulatory factors, patterns of DNA methylation, orchanges in DNA structure. A large number of enhancers from a variety ofdifferent sources are well known in the art and are available as orwithin cloned polynucleotides (from, e.g., depositories such as the ATCCas well as other commercial or individual sources). A number ofpolynucleotides comprising promoters (such as the commonly-used CMVpromoter) also comprise enhancer sequences. Enhancers can be locatedupstream, within, or downstream of coding sequences. The term “Igenhancers” refers to enhancer elements derived from enhancer regionsmapped within the immunoglobulin (Ig) locus (such enhancers include forexample, the heavy chain (mu) 5′ enhancers, light chain (kappa) 5′enhancers, kappa and mu intronic enhancers, and 3′ enhancers (seegenerally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, RavenPress, New York (1993), pages 353-363; and U.S. Pat. No. 5,885,827).

The vector also can comprise a “selectable marker gene.” The term“selectable marker gene,” as used herein, refers to a nucleic acidsequence that allows cells expressing the nucleic acid sequence to bespecifically selected for or against, in the presence of a correspondingselective agent. Suitable selectable marker genes are known in the artand described in, e.g., International Patent Application Publications WO1992/08796 and WO 1994/28143; Wigler et al., Proc. Natl. Acad. Sci. USA,77: 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527(1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981);Colberre-Garapin et al., J. Mol. Biol., 150: 1 (1981); Santerre et al.,Gene, 30: 147 (1984); Kent et al., Science, 237: 901-903 (1987); Wigleret al., Cell, 11: 223 (1977); Szybalska & Szybalski, Proc. Natl. Acad.Sci. USA, 48: 2026 (1962); Lowy et al., Cell, 22: 817 (1980); and U.S.Pat. Nos. 5,122,464 and 5,770,359.

In some embodiments, the vector is an “episomal expression vector” or“episome,” which is able to replicate in a host cell, and persists as anextrachromosomal segment of DNA within the host cell in the presence ofappropriate selective pressure (see, e.g., Conese et al., Gene Therapy,11: 1735-1742 (2004)). Representative commercially available episomalexpression vectors include, but are not limited to, episomal plasmidsthat utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein BarrVirus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4,pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV fromStratagene (La Jolla, Calif.) represent non-limiting examples of anepisomal vector that uses T-antigen and the SV40 origin of replicationin lieu of EBNA1 and oriP.

Other suitable vectors include integrating expression vectors, which mayrandomly integrate into the host cell's DNA, or may include arecombination site to enable the specific recombination between theexpression vector and the host cell's chromosome. Such integratingexpression vectors may utilize the endogenous expression controlsequences of the host cell's chromosomes to effect expression of thedesired protein. Examples of vectors that integrate in a site specificmanner include, for example, components of the flp-in system fromInvitrogen (Carlsbad, Calif.) (e.g., pcDNA™5/FRT), or the cre-loxsystem, such as can be found in the pExchange-6 Core Vectors fromStratagene (La Jolla, Calif.). Examples of vectors that randomlyintegrate into host cell chromosomes include, for example, pcDNA3.1(when introduced in the absence of T-antigen) from Invitrogen (Carlsbad,Calif.), and pCI or pFN10A (ACT) FLEXI™ from Promega (Madison, Wis.).

Viral vectors also can be used. Representative viral expression vectorsinclude, but are not limited to, the adenovirus-based vectors (e.g., theadenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, TheNetherlands)), lentivirus-based vectors (e.g., the lentiviral-based pLP1from Life Technologies (Carlsbad, Calif.)), and retroviral vectors(e.g., the pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, Calif.)).In a preferred embodiment, the viral vector is a lentivirus vector.

The vector comprising the inventive nucleic acid encoding the CAR can beintroduced into a host cell that is capable of expressing the CARencoded thereby, including any suitable prokaryotic or eukaryotic cell.Preferred host cells are those that can be easily and reliably grown,have reasonably fast growth rates, have well characterized expressionsystems, and can be transformed or transfected easily and efficiently.

As used herein, the term “host cell” refers to any type of cell that cancontain the expression vector. The host cell can be a eukaryotic cell,e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell,e.g., bacteria or protozoa. The host cell can be a cultured cell or aprimary cell, i.e., isolated directly from an organism, e.g., a human.The host cell can be an adherent cell or a suspended cell, i.e., a cellthat grows in suspension. Suitable host cells are known in the art andinclude, for instance, DH5α E. coli cells, Chinese hamster ovariancells, monkey VERO cells, COS cells, HEK293 cells, and the like. Forpurposes of amplifying or replicating the recombinant expression vector,the host cell may be a prokaryotic cell, e.g., a DH5α cell. For purposesof producing a recombinant CAR, the host cell can be a mammalian cell.The host cell preferably is a human cell. The host cell can be of anycell type, can originate from any type of tissue, and can be of anydevelopmental stage. In one embodiment, the host cell can be aperipheral blood lymphocyte (PBL), a peripheral blood mononuclear cell(PBMC), or a natural killer (NK). Preferably, the host cell is a naturalkiller (NK) cell. More preferably, the host cell is a T-cell. Methodsfor selecting suitable mammalian host cells and methods fortransformation, culture, amplification, screening, and purification ofcells are known in the art.

The invention provides an isolated host cell which expresses theinventive nucleic acid sequence encoding the CAR described herein. Inone embodiment, the host cell is a T-cell. The T-cell of the inventioncan be any T-cell, such as a cultured T-cell, e.g., a primary T-cell, ora T-cell from a cultured T-cell line, or a T-cell obtained from amammal. If obtained from a mammal, the T-cell can be obtained fromnumerous sources, including but not limited to blood, bone marrow, lymphnode, the thymus, or other tissues or fluids. T-cells can also beenriched for or purified. The T-cell preferably is a human T-cell (e.g.,isolated from a human). The T-cell can be of any developmental stage,including but not limited to, a CD4⁺/CD8⁺ double positive T-cell, a CD4⁺helper T-cell, e.g., Th₁ and Th₂ cells, a CD8⁺ T-cell (e.g., a cytotoxicT-cell), a tumor infiltrating cell, a memory T-cell, a naïve T-cell, andthe like. In one embodiment, the T-cell is a CD8⁺ T-cell or a CD4⁺T-cell. T-cell lines are available from, e.g., the American Type CultureCollection (ATCC, Manassas, Va.), and the German Collection ofMicroorganisms and Cell Cultures (DSMZ) and include, for example, Jurkatcells (ATCC TIB-152), Sup-T1 cells (ATCC CRL-1942), RPMI 8402 cells(DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and derivatives thereof.

In another embodiment, the host cell is a natural killer (NK) cell. NKcells are a type of cytotoxic lymphocyte that plays a role in the innateimmune system. NK cells are defined as large granular lymphocytes andconstitute the third kind of cells differentiated from the commonlymphoid progenitor which also gives rise to B and T lymphocytes (see,e.g., Immunobiology, 5^(th) ed., Janeway et al., eds., GarlandPublishing, New York, N.Y. (2001)). NK cells differentiate and mature inthe bone marrow, lymph node, spleen, tonsils, and thymus. Followingmaturation, NK cells enter into the circulation as large lymphocyteswith distinctive cytotoxic granules. NK cells are able to recognize andkill some abnormal cells, such as, for example, some tumor cells andvirus-infected cells, and are thought to be important in the innateimmune defense against intracellular pathogens. As described above withrespect to T-cells, the NK cell can be any NK cell, such as a culturedNK cell, e.g., a primary NK cell, or an NK cell from a cultured NK cellline, or an NK cell obtained from a mammal. If obtained from a mammal,the NK cell can be obtained from numerous sources, including but notlimited to blood, bone marrow, lymph node, the thymus, or other tissuesor fluids. NK cells can also be enriched for or purified. The NK cellpreferably is a human NK cell (e.g., isolated from a human). NK celllines are available from, e.g., the American Type Culture Collection(ATCC, Manassas, Va.) and include, for example, NK-92 cells (ATCCCRL-2407), NK92MI cells (ATCC CRL-2408), and derivatives thereof.

The inventive nucleic acid sequence encoding a CAR may be introducedinto a cell by “transfection,” “transformation,” or “transduction.”“Transfection,” “transformation,” or “transduction,” as used herein,refer to the introduction of one or more exogenous polynucleotides intoa host cell by using physical or chemical methods. Many transfectiontechniques are known in the art and include, for example, calciumphosphate DNA co-precipitation (see, e.g., Murray E. J. (ed.), Methodsin Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols,Humana Press (1991)); DEAE-dextran; electroporation; cationicliposome-mediated transfection; tungsten particle-facilitatedmicroparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); andstrontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol.,7: 2031-2034 (1987)). Phage or viral vectors can be introduced into hostcells, after growth of infectious particles in suitable packaging cells,many of which are commercially available.

Without being bound to a particular theory or mechanism, it is believedthat by eliciting an antigen-specific response against BCMA, the CARsencoded by the inventive nucleic acid sequence provide for one or moreof the following: targeting and destroying BCMA-expressing cancer cells,reducing or eliminating cancer cells, facilitating infiltration ofimmune cells to tumor site(s), and enhancing/extending anti-cancerresponses. Thus, the invention provides a method of destroying multiplemyeloma cells, which comprises contacting one or more of theaforementioned isolated T-cells or natural killer cells with apopulation of multiple myeloma cells that express BCMA, whereby the CARis produced and binds to BCMA on the multiple myeloma cells and themultiple myeloma cells are destroyed. As discussed herein, multiplemyeloma, also known as plasma cell myeloma or Kahler's disease, is acancer of plasma cells, which are a type of white blood cell normallyresponsible for the production of antibodies (Raab et al., Lancet, 374:324-329 (2009)). Multiple myeloma affects 1-4 per 100,000 people peryear. The disease is more common in men, and for yet unknown reasons istwice as common in African Americans as it is in Caucasian Americans.Multiple myeloma is the least common hematological malignancy (14%) andconstitutes 1% of all cancers (Raab et al., supra). Treatment ofmultiple myeloma typically involves high-dose chemotherapy followed byhematopoietic stem cell transplanatation (allogenic or autologous);however, a high rate of relapse is common in multiple myeloma patientsthat have undergone such treatment. As discussed above, BCMA is highlyexpressed by multiple myeloma cells (see, e.g., Novak et al., supra;Neri et al., supra; Bellucci et al., supra; and Moreaux et al., supra).

One or more isolated T-cells expressing the inventive nucleic acidsequence encoding the anti-BCMA CAR described herein can be contactedwith a population of multiple myeloma cells that express BCMA ex vivo,in vivo, or in vitro. “Ex vivo” refers to methods conducted within or oncells or tissue in an artificial environment outside an organism withminimum alteration of natural conditions. In contrast, the term “invivo” refers to a method that is conducted within living organisms intheir normal, intact state, while an “in vitro” method is conductedusing components of an organism that have been isolated from its usualbiological context. The inventive method preferably involves ex vivo andin vivo components. In this regard, for example, the isolated T-cellsdescribed above can be cultured ex vivo under conditions to express theinventive nucleic acid sequence encoding the anti-BCMA CAR, and thendirectly transferred into a mammal (preferably a human) affected bymultiple myeloma. Such a cell transfer method is referred to in the artas “adoptive cell transfer (ACT),” in which immune-derived cells arepassively transferred into a new recipient host to transfer thefunctionality of the donor immune-derived cells to the new host.Adoptive cell transfer methods to treat various types of cancers,including hematological cancers such as myeloma, are known in the artand disclosed in, for example, Gattinoni et al., Nat. Rev. Immunol.,6(5): 383-393 (2006); June, C H, J. Clin. Invest., 117(6): 1466-76(2007); Rapoport et al., Blood, 117(3): 788-797 (2011); and Barber etal., Gene Therapy, 18: 509-516 (2011)).

The invention also provides a method of destroying Hodgkin's lymphomacells. Hodgkin's lymphoma (formerly known as Hodgkin's disease) is acancer of the immune system that is marked by the presence of amultinucleated cell type called Reed-Sternberg cells. The two majortypes of Hodgkin's lymphoma include classical Hodgkin's lymphoma andnodular lymphocyte-predominant Hodgkin's lymphoma. Hodgkin's lymphomacurrently is treated with radiation therapy, chemotherapy, orhematopoietic stem cell transplantation, with the choice of treatmentdepending on the age and sex of the patient and the stage, bulk, andhistological subtype of the disease. BCMA expression has been detectedon the surface of Hodgkin's lymphoma cells (see, e.g., Chiu et al.,Blood, 109(2): 729-739 (2007)).

When T-cells or NK cells are administered to a mammal, the cells can beallogeneic or autologous to the mammal. In “autologous” administrationmethods, cells (e.g., blood-forming stem cells or lymphocytes) areremoved from a mammal, stored (and optionally modified), and returnedback to the same mammal. In “allogeneic” administration methods, amammal receives cells (e.g., blood-forming stem cells or lymphocytes)from a genetically similar, but not identical, donor. Preferably, thecells are autologous to the mammal.

The T-cells or NK cells desirably are administered to a human in theform of a composition, such as a pharmaceutical composition.Alternatively, the inventive nucleic acid sequence encoding the CAR, ora vector comprising the CAR-encoding nucleic acid sequence, can beformulated into a composition, such as a pharmaceutical composition, andadministered to a human. The inventive pharmaceutical composition cancomprise a population of T-cells of NK cells that express the inventiveCAR. In addition to the inventive nucleic acid sequence, or host cellswhich express the inventive CAR, the pharmaceutical composition cancomprise other pharmaceutically active agents or drugs, such aschemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,vincristine, etc. In a preferred embodiment, the pharmaceuticalcomposition comprises an isolated T-cell or NK cell which expresses theinventive CAR, more preferably a population of T-cells or NK cells whichexpress the inventive CAR.

The inventive T-cells or NK cells can be provided in the form of a salt,e.g., a pharmaceutically acceptable salt. Suitable pharmaceuticallyacceptable acid addition salts include those derived from mineral acids,such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric,and sulphuric acids, and organic acids, such as tartaric, acetic,citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic,and arylsulphonic acids, for example, p-toluenesulphonic acid.

The choice of carrier will be determined in part by the particularinventive nucleic acid sequence, vector, or host cells expressing theCAR, as well as by the particular method used to administer theinventive nucleic acid sequence, vector, or host cells expressing theCAR. Accordingly, there are a variety of suitable formulations of thepharmaceutical composition of the invention. For example, thepharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. A mixture of two or morepreservatives optionally may be used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition.

In addition, buffering agents may be used in the composition. Suitablebuffering agents include, for example, citric acid, sodium citrate,phosphoric acid, potassium phosphate, and various other acids and salts.A mixture of two or more buffering agents optionally may be used. Thebuffering agent or mixtures thereof are typically present in an amountof about 0.001% to about 4% by weight of the total composition.

Methods for preparing administrable (e.g., parenterally administrable)compositions are known to those skilled in the art and are described inmore detail in, for example, Remington: The Science and Practice ofPharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The composition comprising the inventive nucleic acid sequence encodingthe CAR, or host cells expressing the CAR, can be formulated as aninclusion complex, such as cyclodextrin inclusion complex, or as aliposome. Liposomes can serve to target the host cells (e.g., T-cells orNK cells) or the inventive nucleic acid sequence to a particular tissue.Liposomes also can be used to increase the half-life of the inventivenucleic acid sequence. Many methods are available for preparingliposomes, such as those described in, for example, Szoka et al., Ann.Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369.

The composition can employ time-released, delayed release, and sustainedrelease delivery systems such that the delivery of the inventivecomposition occurs prior to, and with sufficient time to cause,sensitization of the site to be treated. Many types of release deliverysystems are available and known to those of ordinary skill in the art.Such systems can avoid repeated administrations of the composition,thereby increasing convenience to the subject and the physician, and maybe particularly suitable for certain composition embodiments of theinvention.

The composition desirably comprises the host cells expressing theinventive nucleic acid sequence encoding a CAR, or a vector comprisingthe inventive nucleic acid sequence, in an amount that is effective totreat or prevent multiple myeloma or Hodgkin's lymphoma. As used herein,the terms “treatment,” “treating,” and the like refer to obtaining adesired pharmacologic and/or physiologic effect. Preferably, the effectis therapeutic, i.e., the effect partially or completely cures a diseaseand/or adverse symptom attributable to the disease. To this end, theinventive method comprises administering a “therapeutically effectiveamount” of the composition comprising the host cells expressing theinventive nucleic acid sequence encoding a CAR, or a vector comprisingthe inventive nucleic acid sequence. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result. Thetherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the CAR to elicit a desired response in the individual. Forexample, a therapeutically effective amount of CAR of the invention isan amount which binds to BCMA on multiple myeloma cells and destroysthem.

Alternatively, the pharmacologic and/or physiologic effect may beprophylactic, i.e., the effect completely or partially prevents adisease or symptom thereof. In this respect, the inventive methodcomprises administering a “prophylactically effective amount” of thecomposition comprising the host cells expressing the inventive nucleicacid sequence encoding a CAR, or a vector comprising the inventivenucleic acid sequence, to a mammal that is predisposed to multiplemyeloma or Hodgkin's lymphoma. A “prophylactically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve a desired prophylactic result (e.g., prevention ofdisease onset).

A typical amount of host cells administered to a mammal (e.g., a human)can be, for example, in the range of one million to 100 billion cells;however, amounts below or above this exemplary range are within thescope of the invention. For example, the daily dose of inventive hostcells can be about 1 million to about 50 billion cells (e.g., about 5million cells, about 25 million cells, about 500 million cells, about 1billion cells, about 5 billion cells, about 20 billion cells, about 30billion cells, about 40 billion cells, or a range defined by any two ofthe foregoing values), preferably about 10 million to about 100 billioncells (e.g., about 20 million cells, about 30 million cells, about 40million cells, about 60 million cells, about 70 million cells, about 80million cells, about 90 million cells, about 10 billion cells, about 25billion cells, about 50 billion cells, about 75 billion cells, about 90billion cells, or a range defined by any two of the foregoing values),more preferably about 100 million cells to about 50 billion cells (e.g.,about 120 million cells, about 250 million cells, about 350 millioncells, about 450 million cells, about 650 million cells, about 800million cells, about 900 million cells, about 3 billion cells, about 30billion cells, about 45 billion cells, or a range defined by any two ofthe foregoing values).

Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment isrepeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and are within the scope ofthe invention. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

The composition comprising the host cells expressing the inventiveCAR-encoding nucleic acid sequence, or a vector comprising the inventiveCAR-encoding nucleic acid sequence, can be administered to a mammalusing standard administration techniques, including oral, intravenous,intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. Thecomposition preferably is suitable for parenteral administration. Theterm “parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. Morepreferably, the composition is administered to a mammal using peripheralsystemic delivery by intravenous, intraperitoneal, or subcutaneousinjection.

The composition comprising the host cells expressing the inventiveCAR-encoding nucleic acid sequence, or a vector comprising the inventiveCAR-encoding nucleic acid sequence, can be administered with one or moreadditional therapeutic agents, which can be coadministered to themammal. By “coadministering” is meant administering one or moreadditional therapeutic agents and the composition comprising theinventive host cells or the inventive vector sufficiently close in timesuch that the inventive CAR can enhance the effect of one or moreadditional therapeutic agents, or vice versa. In this regard, thecomposition comprising the inventive host cells or the inventive vectorcan be administered first, and the one or more additional therapeuticagents can be administered second, or vice versa. Alternatively, thecomposition comprising the inventive host cells or the inventive vectorand the one or more additional therapeutic agents can be administeredsimultaneously. An example of a therapeutic agent that can beco-administered with the composition comprising the inventive host cellsor the inventive vector is IL-2.

Once the composition comprising host cells expressing the inventiveCAR-encoding nucleic acid sequence, or a vector comprising the inventiveCAR-encoding nucleic acid sequence, is administered to a mammal (e.g., ahuman), the biological activity of the CAR can be measured by anysuitable method known in the art. In accordance with the inventivemethod, the CAR binds to BCMA on the multiple myeloma cells, and themultiple myeloma cells are destroyed. Binding of the CAR to BCMA on thesurface of multiple myeloma cells can be assayed using any suitablemethod known in the art, including, for example, ELISA and flowcytometry. The ability of the CAR to destroy multiple myeloma cells canbe measured using any suitable method known in the art, such ascytotoxicity assays described in, for example, Kochenderfer et al., J.Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. ImmunologicalMethods, 285(1): 25-40 (2004). The biological activity of the CAR alsocan be measured by assaying expression of certain cytokines, such asCD107a, IFNγ, IL-2, and TNF.

One of ordinary skill in the art will readily appreciate that theinventive CAR-encoding nucleic acid sequence can be modified in anynumber of ways, such that the therapeutic or prophylactic efficacy ofthe CAR is increased through the modification. For instance, the CAR canbe conjugated either directly or indirectly through a linker to atargeting moiety. The practice of conjugating compounds, e.g., the CAR,to targeting moieties is known in the art. See, for instance, Wadwa etal., J. Drug Targeting 3: 111 (1995), and U.S. Pat. No. 5,087,616.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the expression pattern of BCMA in human cells.

Quantitative polymerase chain reaction (qPCR) was performed on a panelof cDNA samples from a wide range of normal tissues included in theHuman Major Tissue qPCR panel II (Origine Technologies, Rockville, Md.)using a BCMA-specific primer and probe set (Life Technologies, Carlsbad,Calif.). cDNA from cells of a plasmacytoma that was resected from apatient with advanced multiple myeloma was analyzed as a positivecontrol. RNA was extracted from the plasmacytoma cells with an RNeasymini kit (Qiagen, Inc., Valencia, Calif.), and cDNA was synthesizedusing standard methods. A standard curve for the BCMA qPCR was createdby diluting a plasmid that encoded the full-length BCMA cDNA (OrigineTechnologies, Rockville, Md.) in carrier DNA. The qPCR accuratelydetected copy numbers from 10² to 10⁹ copies of BCMA per reaction. Thenumber of β-actin cDNA copies in the same tissues was quantitated with aTaqman β-actin primer and probe kit (Life Technologies, Carlsbad,Calif.). A β-actin standard curve was created by amplifying serialdilutions of a β-actin plasmid. All qPCR reactions were carried out onthe Roche LightCycler480 machine (Roche Applied Sciences, Indianapolis,Ind.).

The results of the qPCR analysis are depicted in FIGS. 1A and 1B. 93%percent of the cells from the plasmacytoma sample were plasma cells asdetermined by flow cytometry. BCMA expression in the plasmacytoma samplewas dramatically higher than BCMA expression in any other tissue. BCMAcDNA was detected in several hematologic tissues, such as peripheralblood mononuclear cells (PBMC), bone marrow, spleen, lymph node, andtonsil. Low levels of BCMA cDNA were detected in most gastrointestinalorgans, such as duodenum, rectum, and stomach. BCMA expression ingastrointestinal organs may be the result of plasma cells and B-cellspresent in gut-associated lymphoid tissues such as the lamina propriaand Peyer's Patches (see, e.g., Brandtzaeg, ImmunologicalInvestigations, 39(4-5): 303-355 (2010)). Low levels of BCMA cDNA alsowere detected in the testis and the trachea. The low levels of BCMA cDNAdetected in the trachea may be due to the presence of plasma cells inthe lamina propria of the trachea (see, e.g., Soutar, Thorax,31(2):158-166 (1976)).

The expression of BCMA on the surface of various cell types was furthercharacterized using flow cytometry (see FIGS. 2A-2L), including multiplemyeloma cell lines H929, U266, and RPMI8226. The multiple myeloma celllines H929, U266, and RPMI8226 all expressed cell surface BCMA. Incontrast, the sarcoma cell line TC71, the T-cell leukemia line CCRF-CEM,and the kidney cell line 293T-17 did not express cell surface BCMA.Primary CD34⁺ hematopoietic cells, primary small airway epithelialcells, primary bronchial epithelial cells, and primary intestinalepithelial cells all lacked cell surface BCMA expression.

The results of this example demonstrate that BCMA is expressed on thesurface of multiple myeloma cells, and it has a restricted expressionpattern in normal tissues.

EXAMPLE 2

This example describes the construction of the inventive nucleic acidsequence encoding anti-BCMA chimeric antigen receptors (CARs).

Antibody sequences of two mouse-anti-human-BCMA antibodies designated as“C12A3.2” and “C11D5.3” were obtained from International PatentApplication Publication WO 2010/104949 (Kalled et al.). The amino acidsequences of the heavy chain variable regions and light chain variableregions of these antibodies were used to design single chain variablefragments (scFvs) having the following general structure:

-   -   light chain variable region—linker—heavy chain variable region.

The linker had the following amino acid sequence: GSTSGSGKPGSGEGSTKG(SEQ ID NO: 7) (see, e.g., Cooper et al., Blood, 101(4): 1637-1644(2003)).

DNA sequences encoding two chimeric antigen receptors were designed,each of which contained the following elements from 5′ to 3′: the CD8αsignal sequence, the aforementioned anti-BCMA scFv, hinge andtransmembrane regions of the human CD8α molecule, the cytoplasmicportion of the CD28 molecule, and the cytoplasmic portion of the CD3ζmolecule. A schematic of these CAR-encoding nucleic acid sequences isset forth in FIG. 3A. The CARs incorporating variable regions fromC12A3.2 and C11D5.3 were designated anti-bcma1 and anti-bcma2,respectively.

DNA sequences encoding five additional chimeric antigen receptors basedon the above-described anti-bcma2 CAR were designed, each of whichcontained different signal sequences and T-cell activation domains. Inthis respect, 8ss-anti-bcma2 CAR contained the following elements from5′ to 3: the CD8α signal sequence, scFv, hinge and transmembrane regionsof the human CD8α molecule, the cytoplasmic portion of the CD28molecule, and the cytoplasmic portion of the CD3ζ molecule. TheG-anti-bcma2 CAR contained the following elements from 5′ to 3′: thehuman GM-CSF receptor signal sequence, scFv, hinge and transmembraneregions of the human CD8α molecule, the cytoplasmic portion of the CD28molecule, and the cytoplasmic portion of the CD3ζ molecule. Theanti-bcma2-BB CAR contained the following elements from 5′ to 3′: theCD8α signal sequence, scFv, hinge and transmembrane regions of the humanCD8α molecule, the cytoplasmic portion of the 4-1BB molecule, and thecytoplasmic portion of the CD3ζ molecule. The anti-bcma2-OX40 CARcontained the following elements from 5′ to 3′: the CD8α signalsequence, scFv, hinge and transmembrane regions of the human CD8αmolecule, the cytoplasmic portion of the OX40 molecule (see, e.g., Latzaet al., European Journal of Immunology, 24: 677-683 (1994)), and thecytoplasmic portion of the CD3ζ molecule. The anti-bcma2-BBOX40contained the following elements from 5′ to 3′: the CD8α signalsequence, scFv, hinge and transmembrane regions of the human CD8αmolecule, the cytoplasmic portion of the 4-1BB molecule, the cytoplasmicregion of the OX40 molecule, and the cytoplasmic portion of the CD3ζmolecule. The elements present in each of the seven CAR sequences areset forth in Table 1.

TABLE 1 Hinge and Intracellular SEQ ID NO Signal Transmembrane T-cellSignaling CAR (amino acid) Sequence Regions Domain anti-bcma1 4 HumanCD8α Human CD8α CD28 CD3ζ anti-bcma2 5 Human CD8α Human CD8α CD28 CD3ζG-Anti-bcma2 8 GM-CSF Human CD8α CD28 receptor CD3ζ 8ss-anti-bcma2 9Human CD8α Human CD8α CD28 CD3ζ anti-bcma2-BB 10 Human CD8α Human CD8α4-1BB CD3ζ anti-bcma2-OX40 11 Human CD8α Human CD8α OX40 CD3ζanti-bcma2-BBOX40 12 Human CD8α Human CD8α 4-1BB OX40 CD3ζ

The sequences used for CD8α, CD28, CD3ζ, 4-1BB (CD137), and OX40 (CD134)were obtained from the publicly available National Center forBiotechnology Information (NCBI) database.

The CAR-encoding nucleic acid sequences were generated using methodsknown in the art, such as those described in, for example, Kochenderferet al., J. Immunology, 32(7): 689-702 (2009), and Zhao et al., J.Immunology, 183(9): 5563-5574 (2009). The nucleic acid sequence encodingeach CAR was codon optimized and synthesized using GeneArt™ technology(Life Technologies, Carlsbad, Calif.) with appropriate restrictionsites.

The sequences encoding the anti-bcma1 and anti-bcma2 CARs were ligatedinto a lentiviral vector plasmid designatedpRRLSIN.cPPT.MSCV.coDMF5.oPRE (see, e.g., Yang et al., J. Immunotherapy,33(6): 648-658 (2010)). The coDMF5 portion of this vector was replacedwith the CAR-encoding nucleic acid sequences using standard methods. Thetwo resulting anti-BCMA CAR vectors were denotedpRRLSIN.cPPT.MSCV.anti-bcma1.oPRE and pRRLSIN.cPPT.MSCV.anti-bcma2.oPRE.A negative-control CAR containing the SP6 scFv that recognizes thehapten 2,4,6-trinitrophenyl also was constructed (see, e.g., Gross etal., Proc. Natl. Acad. Sci. USA, 86(24): 10024-10028 (1989)). This CARwas referred to as SP6. The SP6 CAR was cloned into the same lentiviralvector as the anti-BCMA CARs and contained the same signaling domains asanti-bcma1 and anti-bcma2. Supernatant containing lentiviruses encodingeach CAR was produced by the protocol described in Yang et al., supra.Specifically, 293T-17 cells (ATCC CRL-11268) were transfected with thefollowing plasmids: pMDG (encoding the vesicular stomatitis virusenvelope protein), pMDLg/pRRE (encoding HIV Gag and Pol proteins),pRSV-Rev (encoding RSV Rev protein), and plasmids encoding the anti-bcmaCARs (see, e.g., Yang et al., supra).

The sequences encoding the G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB,anti-bcma2-OX40, and anti-bcma2-BBOX40 CARs were each ligated into agammaretroviral vector plasmid designated MSGV (mouse stem cellvirus-based splice-gag vector) using standard methods, such as thosedescribed in, e.g., Hughes et al., Human Gene Therapy, 16: 457-472(2005). After the CAR-encoding gammaretroviral plasmids were generated,replication incompetent retroviruses with the RD114 envelope wereproduced by transient transfection of 293-based packaging cells asdescribed in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702(2009).

The replication-incompetent lentiviruses and retroviruses encoding theabove-described CARs were used to transduce human T-cells. Foranti-bcma1 and anti-bcma2, T-cells were cultured as described previously(see, e.g., Kochenderfer et al., J. Immunotherapy, 32(7): 689-702(2009)) and were stimulated with the anti-CD3 monoclonal antibody OKT3(Ortho-Biotech, Horsham, Pa.) in AIM V™ medium (Life Technologies,Carlsbad, Calif.) containing 5% human AB serum (Valley Biomedical,Winchester, Va.) and 300 international units (IU)/mL of interleukin-2(Novartis Diagnostics, Emeryville, Calif.). Thirty-six hours after thecultures were started, the activated T-cells were suspended inlentiviral supernatant with protamine sulfate and 300 IU/mL IL-2. Thecells were centrifuged for 1 hour at 1200×g. The T-cells were thencultured for three hours at 37° C. The supernatant was then diluted 1:1with RPMI medium (Mediatech, Inc., Manassas, Va.)+10% fetal bovine serum(Life Technologies, Carlsbad, Calif.) and IL-2. The T-cells werecultured in the diluted supernatant overnight, and then returned toculture in AIM V™ medium (Life Technologies, Carlsbad, Calif.) plus 5%human AB serum with IL-2. T-cells were stained with biotin-labeledpolyclonal goat anti-mouse-F(ab)₂ antibodies (Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa.) to detect the anti-BCMA CARs. Highlevels of cell surface expression of the anti-bcma1 CAR, the anti-bcma2CAR, and the SP6 CAR on the transduced T-cells were observed, as shownin FIGS. 3B-3D.

For the G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40,and anti-bcma2-BBOX40 CARs, peripheral blood mononuclear cells weresuspended at a concentration of 1×10⁶ cell per mL in T-cell mediumcontaining 50 ng/mL of the anti-CD3 monoclonal antibody OKT3 (Ortho,Bridgewater, N.J.) and 300 IU/mL of IL-2. RETRONECTIN™ polypeptide(Takara Bio Inc., Shiga, Japan), which is a recombinant polypeptide ofhuman fibronectin fragments that binds viruses and cell surfaceproteins, was dissolved at a concentration of 11 μg/mL in phosphatebuffered saline (PBS) solution, and two mL of the RETRONECTIN™polypeptide in PBS solution were added to each well ofnontissue-culture-coated 6 well plates (BD Biosciences, Franklin Lakes,N.J.). The plates were incubated for two hours at room temperature (RT).After the incubation, the RETRONECTIN™ solution was aspirated, and 2 mLof a blocking solution consisting of Hanks' balanced salt solution(HBSS) plus 2% bovine serum albumin (BSA) were added to eachRETRONECTIN™-coated well. The plates were incubated for 30 minutes atroom temperature (RT). The blocking solution was aspirated, and thewells were rinsed with a solution of HBSS+2.5%(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). Retroviralsupernatant was rapidly thawed and diluted 1:1 in T-cell media, and twomL of the diluted supernatant were then added to eachRETRONECTIN™-coated well. After addition of the supernatants, the plateswere centrifuged at 2000×g for 2 hours at 32° C. The supernatant wasthen aspirated from the wells, and 2×10⁶ T-cells that had been culturedwith OKT3 antibody and IL-2 for 2 days were added to each well. When theT-cells were added to the retrovirus-coated plates, the T-cells weresuspended at a concentration of 0.5×10⁶ cells per mL in T-cell mediumplus 300 IU/mL of IL-2. After the T-cells were added to each well, theplates were centrifuged for 10 minutes at 1000×g. The plates wereincubated at 37° C. overnight. The transduction was repeated the nextday. After an 18-24 hour incubation, the T-cells were removed from theplates and suspended in fresh T-cell medium with 300 IU/mL of IL-2 at aconcentration of 0.5×10⁶ cells per mL and cultured at 37° C. and 5% CO₂.High levels of cell surface expression of anti-bcma2-BBOX40,anti-bcma2-BB, and 8ss-anti-bcma2 on the transduced T-cells wereobserved.

The results of this example demonstrate a method of producing theinventive CAR-encoding nucleic acid sequence, and methods of expressingthe CAR on the surface of T-cells.

EXAMPLE 3

This example describes a series of experiments used to determine thespecificity of the inventive CAR for BCMA.

Cells

NCI-H929, U266, and RPMI8226 are all BCMA+ multiple myeloma cell linesthat were obtained from ATCC (ATCC Nos. CRL-9068, TIB-196, and CCL-155,respectively). A549 (ATCC No. CCL-185) is a BCMA-negative lung cancercell line. TC71 is a BCMA-negative sarcoma cell line. CCRF-CEM is aBCMA-negative T-cell line (ATCC No. CCL-119). BCMA-K562 are K562 cells(ATCC No. CCL-243) that have been transduced with a nucleic acidsequence encoding full-length BCMA. NGFR-K562 are K562 cells that havebeen transduced with the gene encoding low-affinity nerve growth factor(see, e.g., Kochenderfer et al., J. Immunotherapy., 32(7):689-702(2009)). Peripheral blood lymphocytes (PBL) from three patients withmultiple myeloma (i.e., Myeloma Patient 1 through 3) were used, as werePBL from three other subjects: Donor A, Donor B, and Donor C. Donors Athrough C all had melanoma. CD34+ primary cells were obtained from threenormal healthy donors. A sample of plasmacytoma cells was obtained fromMyeloma patient 1, and a sample of bone marrow was obtained from MyelomaPatient 3. All of the human samples mentioned above were obtained frompatients enrolled in IRB-approved clinical trials at the National CancerInstitute. The following primary human epithelial cells were obtainedfrom Lonza, Inc. (Basel, Switzerland): small airway epithelial cells,bronchial epithelial cells, and intestinal epithelial cells.

Interferon-γ and TNF ELISA

BCMA-positive or BCMA-negative cells were combined with CAR-transducedT-cells in duplicate wells of a 96 well round bottom plate (Corning LifeSciences, Lowell, Mass.) in AIM V™ medium (Life Technologies, Carlsbad,Calif.)+5% human serum. The plates were incubated at 37° C. for 18-20hours. Following the incubation, ELISAs for IFNγ and TNF were performedusing standard methods (Pierce, Rockford, Ill.).

T-cells transduced with the anti-bcma1 or anti-bcma2 CARs produced largeamounts of IFNγ when they were cultured overnight with theBCMA-expressing cell line BCMA-K562, but the CAR-transduced T-cells onlyproduced background levels of IFNγ when they were cultured with thenegative control cell line NGFR-K562, as indicated in Table 2 (all unitsare pg/mL IFNγ).

TABLE 2 BCMA-Expressing Targets** BCMA-Negative Targets T-cells EffectorCells* BCMA-K562 H929 RPMI-8226 NGFR-K562 CCRF-CEM A549 TC71 293T Aloneanti-bcma1 15392 11306 5335 76 76 52 65 54 112 anti-bcma2 25474 2312010587 62 67 32 31 28 41 SP6 32 60 149 27 28 21 361 73 27 Untransduced<12 <12 <12 <12 <12 <12 <12 12 <12 Targets Alone <12 <12 <12 <12 <12 <12<12 13 *Effector cells were T-cells from a patient with multiple myeloma(Myeloma Patient 2). The T-cells were transduced with the indicated CARor left untransduced. **The indicated target cells were combined withthe effector cells for an overnight incubation and an IFNγ ELISA wasperformed.

T-cells expressing the 8ss-anti-bcma2, anti-bcma2-BB, andanti-bcma2-OX40 CARs produced IFNγ specifically in response to BCMA+target cells when T-cells and target cells were cocultured overnight, asindicated in Table 3 (all units are pg/mL of IFNγ).

TABLE 3 BCMA-Positive Targets BCMA-Negative Targets Effector CellsBCMA-K562 RPMI-8226 NGFR-K562 CCRF-CEM A549 T-cells Aloneanti-bcma2-OX40 17704 4875 42 44 24 40 anti-bcma2-BB 25304 8838 404 602350 706 8ss-anti-bcma2 9671 2168 100 120 49 171 Untransduced <12 57 1517 <12 20

T-cells transduced with anti-BCMA CARs produced large amounts of IFNγwhen they were cultured overnight with BCMA-expressing multiple myelomacell lines. In contrast, the anti-BCMA CARs produced much lower amountsof IFNγ when they were cultured with a variety of BCMA-negative celllines. Compared with T-cells transduced with the anti-bcma1 CAR, T-cellstransduced with the anti-bcma2 CAR and variants thereof (i.e.,8ss-anti-bcma2, anti-bcma2-BB, and anti-bcma2-OX40) produced more IFNγwhen cultured with BCMA-positive cells and less IFNγ when cultured withBCMA-negative cells.

T-cells transduced with the anti-bcma2 CAR variants produced TNFspecifically in response to BCMA+ target cells when T-cells and targetcells were cocultured overnight, as indicated in Table 4 (all units arepg/mL of tumor necrosis factor (TNF)).

TABLE 4 BCMA-Positive Targets BCMA-Negative Targets Effector CellsBCMA-K562 RPMI-8226 NGFR-K562 CCRF-CEM A549 T-cells Aloneanti-bcma2-OX40 4913 3406 <40 47 <40 74 anti-bcma2-BB 6295 2723 56 16489 252 8ss-anti-bcma2 5340 1354 <40 121 <40 191 Untransduced <40 <40 47<40 <40 <40

Because the T-cells transduced with the anti-bcma2 CAR and variantsthereof exhibited slightly stronger and more specific recognition ofBCMA-expressing cells than T-cells transduced with the anti-bcma1 CAR,only the anti-bcma2 CAR and anti-bcma2 CAR variants were used in thefollowing experiments.

CD107a Assay

Two populations of T-cells were prepared in two separate tubes. One tubecontained BCMA-K562 cells, and the other tube contained NGFR-K562 cells.Both tubes also contained T-cells transduced with the anti-bcma2 CAR andanti-bcma2 CAR variants, 1 mL of AIM V™ medium (Life Technologies,Carlsbad, Calif.)+5% human serum, a titrated concentration of ananti-CD107a antibody (eBioscience, Inc., San Diego, Calif.; cloneeBioH4A3), and 1 μL of Golgi Stop (BD Biosciences, Franklin Lakes,N.J.). All tubes were incubated at 37° C. for four hours and thenstained for expression of CD3, CD4, and CD8.

CAR-transduced T-cells from three different subjects upregulated CD107aspecifically in response to stimulation with BCMA-expressing targetcells (see FIGS. 4A-4C). This indicates the occurrence of BCMA-specificdegranulation of the T-cells, which is a prerequisite forperforin-mediated cytotoxicity (see, e.g., Rubio et al., NatureMedicine, 9(11): 1377-1382 (2003)). In addition, T-cells expressing theanti-bcma2 CAR variants 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40degranulated in a BCMA-specific manner when stimulated with target cellsin vitro as shown in FIGS. 5A-5D.

Intracellular Cytokine Staining Assay (ICCS)

A population of BCMA-K562 cells and a population of NGFR-K562 cells wereprepared in two separate tubes as described above. Both tubes alsocontained T-cells transduced with the anti-bcma2 CAR from MyelomaPatient 2, 1 mL of AIM V medium (Life Technologies, Carlsbad, Calif.)+5%human serum, and 1 μL of Golgi Stop (BD Biosciences, Franklin Lakes,N.J.). All tubes were incubated at 37° C. for six hours. The cells weresurface-stained with anti-CD3, anti-CD4, and anti-CD8 antibodies. Thecells were permeabilized, and intracellular staining was conducted forIFNγ (BD Biosciences, Franklin Lakes, N.J., clone B27), IL-2 (BDBiosciences, Franklin Lakes, N.J., clone MQ1-17H12), and TNF (BDBiosciences, Franklin Lakes, N.J., clone MAb11) by following theinstructions of the Cytofix/Cytoperm kit (BD Biosciences, FranklinLakes, N.J.).

Large populations of T-cells transduced with the anti-bcma2 CAR fromMyeloma Patient 2 specifically produced the cytokines IFNγ, IL-2, andTNF in a BCMA-specific manner after the six-hour stimulation withBCMA-expressing target cells, as shown in FIGS. 6A-6C.

Proliferation Assays

The ability of T-cells transduced with the anti-bcma2 CAR to proliferatewhen stimulated with BCMA-expressing target cells was assessed.Specifically, 0.5×10⁶ irradiated BCMA-K562 cells or 0.5×10⁶ irradiatedNGFR-K562 cells were co-cultured with 1×10⁶ total T-cells that had beentransduced with either the anti-bcma2 CAR or the SP6 CAR. The T-cellswere labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE)(Life Technologies, Carlsbad, Calif.) as described in Mannering et al.,J. Immunological Methods, 283(1-2): 173-183 (2003). The medium used inthe co-cultures was AIM V™ medium (Life Technologies, Carlsbad,Calif.)+5% human AB serum. IL-2 was not added to the medium. Four daysafter initiation, the live cells in each co-culture were counted withtrypan blue for dead cell exclusion. Flow cytometry was then performedby staining T-cells with polyclonal biotin-labeled goat-anti-human BCMAantibodies (R&D Systems, Minneapolis, Minn.) followed by streptavidin(BD Biosciences, Franklin Lakes, N.J.), anti-CD38 antibody (eBioscience,Inc., San Diego, Calif.), and anti-CD56 antibody (BD Biosciences,Franklin Lakes, N.J.). Flow cytometry data analysis was performed byusing FlowJo software (Tree Star, Inc., Ashland, Oreg.).

T-cells that expressed the anti-bcma2 CAR exhibited a greater dilutionof CFSE when cultured with the BCMA-K562 cells than when cultured withnegative control NGFR-K562 cells, as shown in FIG. 7A. These resultsindicate that T-cells transduced with the anti-bcma2 CAR specificallyproliferated when stimulated with BCMA-expressing target cells. Incontrast, there was no significant difference in CFSE dilution whenT-cells expressing the SP6 CAR were cultured with either BCMA-K562target cells or NGFR-K562 target cells (see FIG. 7B), which demonstratesa lack of BCMA-specific proliferation by T-cells expressing the SP6 CAR.

At the beginning of the proliferation assays, 0.8×10⁶ T-cells expressingthe anti-bcma2 CAR were cultured with either BCMA-K562 cells orNGFR-K562 cells. After 4 days of culture, 2.7×10⁶ T-cells expressing theanti-bcma2 CAR were present in the cultures containing BCMA-K562 cellswhile only 0.6×10⁶ T-cells expressing the anti-bcma2 CAR were present inthe cultures containing NGFR-K562 cells. This BCMA-specific increase inthe absolute number of T-cells expressing the anti-bcma2 CAR indicatesthat these T-cells proliferated in response to BCMA.

The results of this example demonstrate that T-cells expressing theinventive CAR exhibit BCMA-specific cytokine production, degranulation,and proliferation.

EXAMPLE 4

This example demonstrates that T-cells expressing the inventiveanti-BCMA CAR can destroy multiple myeloma cell lines.

Cytotoxicity assays were performed to determine whether T-cellstransduced with the anti-bcma2 CAR described in Examples 2 and 3 coulddestroy BCMA-expressing multiple myeloma (MM) cell lines. Specifically,the cytotoxicity of target cells was measured by comparing the survivalof BCMA-expressing target cells (i.e., multiple myeloma cell lines H929and RPMI8226) relative to the survival of negative control CCRF-CEMcells using an assay described in, e.g., Kochenderfer et al., J.Immunotherapy, 32(7): 689-702 (2009), and Hermans et al., J.Immunological Methods, 285(1): 25-40 (2004).

Approximately 50,000 BCMA-expressing target cells and 50,000 CCRF-CEMcells were combined in the same tubes with different numbers ofCAR-transduced T-cells. CCRF-CEM negative control cells were labeledwith the fluorescent dye5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR)(Life Technologies, Carlsbad, Calif.), and BCMA-expressing target cellswere labeled with CFSE. In all experiments, the cytotoxicity of effectorT-cells that were transduced with the anti-bcma2 CAR was compared to thecytotoxicity of negative control effector T-cells from the same subjectthat were transduced with the SP6 CAR. Co-cultures were established insterile 5 mL test tubes (BD Biosciences, Franklin Lakes, N.J.) induplicate at the following T-cell:target cell ratios: 20.0:1, 7:1, 2:1,and 0.7:1. The cultures were incubated for four hours at 37° C.Immediately after the incubation, 7-amino-actinomycin D (7AAD; BDBiosciences, Franklin Lakes, N.J.) was added. The percentages of liveBCMA-expressing target cells and live CCRF-CEM negative control cellswere determined for each T-cell/target cell co-culture.

For each T-cell/target cell co-culture, the percent survival ofBCMA-expressing target cells relative to the CCRF-CEM negative controlcells was determined by dividing the percent BCMA-expressing cells bythe percent CCRF-CEM negative control cells. The corrected percentsurvival of BCMA-expressing target cells was calculated by dividing thepercent survival of BCMA-expressing target cells in each T-cell/targetcell co-culture by the ratio of the percent BCMA-expressing targetcells:percent CCRF-CEM negative control cells in tubes containing onlyBCMA-expressing target cells and CCRF-CEM negative control cells withouteffector T-cells. This correction was necessary to account for variationin the starting cell numbers and for spontaneous target cell death.Cytotoxicity was calculated as follows:% cytotoxicity of BCMA-expressing target cells=100-corrected % survivalof BCMA-expressing target cells

The results of the cytotoxicity assay are shown in FIGS. 7C and 7D.T-cells transduced with the anti-bcma2 CAR specifically killed theBCMA-expressing multiple myeloma cell lines H929 and RPMI8226. Incontrast, T-cells transduced with the SP6 CAR exhibited much lowerlevels of cytotoxicity against these cell lines.

The results of this example demonstrate that the inventive nucleic acidsequence encoding an anti-BCMA CAR can be used in a method of destroyingmultiple myeloma cell lines.

EXAMPLE 5

This example demonstrates that T-cells expressing the inventiveanti-BCMA CAR can destroy primary multiple myeloma cells.

The primary multiple myeloma cells described in Example 2 were evaluatedfor BCMA expression, as well as BCMA-specific cytokine production,degranulation, and proliferation using the methods described above.

Cell surface BCMA expression was detected on four primary multiplemyeloma samples, as well as on primary bone marrow multiple myelomacells from Myeloma Patient 3 (see FIG. 8A). BCMA-expressing plasma cellsmade up 40% of the cells in the bone marrow sample from Myeloma Patient3. Allogeneic T-cells transduced with the anti-bcma2 CAR from Donor Cproduced IFNγ after co-culture with the unmanipulated bone marrow cellsof Myeloma Patient 3, as shown in FIG. 8B. Anti-bcma2 CAR-transducedT-cells from the same allogeneic donor produced much less IFNγ when theywere cultured with peripheral blood mononuclear cell (PBMC) from MyelomaPatient 3. In addition, SP6-CAR-transduced T-cells from Donor C did notspecifically recognize the bone marrow of Myeloma Patient 3. It has beenpreviously reported that normal PBMC does not contain cells that expressBCMA (see, e.g., Ng et al., J. Immunology, 173(2): 807-817 (2004)). Toconfirm this observation, PBMC of Patient 3 was assessed for BCMAexpression by flow cytometry. PBMC of Patient 3 did not containBCMA-expressing cells, aside from a small population of CD56+CD38^(high)cells that made up approximately 0.75% of the PBMC. This populationpossibly consisted of circulating multiple myeloma cells.

A plasmacytoma resected from Myeloma Patient 1 consisted of 93% plasmacells, and these primary plasma cells expressed BCMA, as shown in FIG.8C. T-cells from Myeloma Patient 2 produced IFNγ when cultured with theallogeneic, unmanipulated plasmacytoma cells of Myeloma Patient 1.T-cells from Myeloma Patient 2 did not produce significant amounts ofIFNγ when cultured with PBMC from Myeloma patient 1. T-cells fromMyeloma Patient 2 that were transduced with the SP6 CAR did not producesignificant amounts of IFNγ when they were cultured with eitherplasmacytoma cells or PBMC from Myeloma Patient 1. The PBMC of MyelomaPatient 1 did not express BCMA as measured by flow cytometry.

T-cells of Myeloma Patient 1, who had received eight prior cycles ofmyeloma therapy, were successfully cultured and transduced with alentivirus vector encoding the anti-bcma2 CAR. Eight days after thecultures were initiated, expression of the anti-bcma2 CAR was detectedon 65% of the T-cells. The T-cells from Myeloma Patient 1 expressing theanti-bcma2 CAR produced IFNγ specifically in response to autologousplasmacytoma cells (FIG. 8D). T-cells from Myeloma Patient 1 expressingthe SP6 CAR did not recognize autologous plasmacytoma cells. T-cellsexpressing the anti-bcma2 CAR and T-cells expressing the SP6 CAR did notrecognize autologous PBMC. T-cells from Myeloma Patient 1 expressing theanti-bcma2 CAR also specifically killed autologous plasmacytoma cells atlow effector to target ratios. In contrast, T-cells from Myeloma Patient1 expressing the SP6 CAR exhibited low levels of cytotoxicity againstautologous plasmacytoma cells (FIG. 8E).

The results of this example demonstrate that the inventive anti-BCMA CARcan be used in a method of destroying primary multiple myeloma cells.

EXAMPLE 6

This example demonstrates that T-cells expressing the inventiveanti-BCMA CARs can destroy established tumors in mice.

Immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, JacksonLaboratory) were injected intradermally with 8×10⁶ RPMI8226 cells.Tumors were allowed to grow for 17 to 19 days, and then the micereceived intravenous infusions of 8×10⁶ human T-cells that weretransduced with either the anti-bcma2 CAR or the SP6 CAR. Tumors weremeasured with calipers every 3 days. The longest length and the lengthperpendicular to the longest length were multiplied to obtain the tumorsize (area) in mm². When the longest length reached 15 mm, mice weresacrificed. Animal studies were approved by the National CancerInstitute Animal Care and Use Committee.

The results of this example are shown in FIGS. 9A and 9B. At around day6, mice treated with anti-bcma2-transduced T-cells showed a reduction intumor size, and tumors were eradicated at day 15. In addition, all micetreated with anti-bcma2-transduced T-cells survived out to 30 days postT-cell infusion.

The results of this example demonstrate that the inventive anti-BMCA CARcan destroy multiple myeloma cells in vivo.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A chimeric antigen receptor comprising: (a)an antigen binding domain targeting B-Cell Maturation Antigen (BCMA),wherein the antigen binding domain comprises an antibody or antigenbinding fragment thereof; (b) a transmembrane domain; (c) a 4-1BBsignaling domain; and (d) a CD3ζ signaling domain.
 2. The chimericantigen receptor of claim 1, wherein the antigen binding domaincomprises a single chain variable fragment (scFv).
 3. The chimericantigen receptor of claim 2, wherein the amino acid sequence of the scFvcomprises the (i) heavy chain complementarity determining region (CDR)1,(ii) heavy chain CDR2, (iii) heavy chain CDR3, (iv) light chain CDR1,(v) light chain CDR2, and (vi) light chain CDR3 of one amino acidsequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, and SEQ ID NO:
 12. 4. The chimeric antigen receptor of claim 1,wherein the transmembrane domain is a human CD28 transmembrane domain.5. The chimeric antigen receptor of claim 1, wherein the transmembranedomain is a human CD8α transmembrane domain.
 6. The chimeric antigenreceptor of claim 1, wherein the chimeric antigen receptor furthercomprises a hinge domain.
 7. The chimeric antigen receptor of claim 6,wherein the hinge domain is a human CD8α hinge domain.
 8. The chimericantigen receptor of claim 6, wherein the hinge domain is a human CD28hinge domain.
 9. The chimeric antigen receptor of claim 6, wherein theantigen binding domain comprises an scFv, the hinge domain is a humanCD8α hinge domain, and the transmembrane domain is a human CD8αtransmembrane domain.
 10. The chimeric antigen receptor of claim 6,wherein the antigen binding domain comprises an scFv, the hinge domain ahuman CD28 hinge domain, and the transmembrane domain is a human CD28transmembrane domain.
 11. The chimeric antigen receptor of claim 6,wherein the antigen binding domain comprises an scFv, the hinge domainis a human CD8α hinge domain, and the transmembrane domain is a humanCD28 transmembrane domain.
 12. The chimeric antigen receptor of claim 6,wherein the antigen binding domain comprises an scFv, the hinge domainis a human CD28 hinge domain, and the transmembrane domain is a humanCD8α transmembrane domain.
 13. The chimeric antigen receptor of claim 1,wherein the chimeric antigen receptor further comprises a signalsequence.
 14. The chimeric antigen receptor of claim 13, wherein thesignal sequence is a granulocyte-macrophage colony-stimulating factor(GM-CSF) receptor signal sequence or a CD8α signal sequence.
 15. Thechimeric antigen receptor of claim 13, wherein the signal sequence is aCD8α signal sequence.
 16. The chimeric antigen receptor of claim 1,wherein the CAR comprises a CD8α signal sequence, an scFv antigenbinding domain, a human CD8α hinge domain, a human CD8α transmembranedomain, a human 4-1BB signaling domain, and a human CD3 signalingdomain.
 17. The chimeric antigen receptor of claim 1, wherein the CARcomprises a CD8α signal sequence, an scFv antigen binding domain, ahuman CD28 hinge domain, a human CD28 transmembrane domain, a human4-1BB signaling domain, and a human CD3 signaling domain.
 18. Thechimeric antigen receptor of claim 1, wherein the CAR comprises a CD8αsignal sequence, an scFv antigen binding domain, a human CD8α hingedomain, a human CD28 transmembrane domain, a human 4-1BB signalingdomain, and a human CD3 signaling domain.
 19. The chimeric antigenreceptor of claim 1, wherein the CAR comprises a CD8α signal sequence,an scFv antigen binding domain, a human CD28 hinge domain, a human CD8αtransmembrane domain, a human 4-1BB signaling domain, and a human CD3signaling domain.
 20. A nucleic acid sequence encoding the chimericantigen receptor of claim
 1. 21. The nucleic acid sequence of claim 20,wherein the nucleic acid sequence encodes an amino acid sequence that ismore than 80% identical to the amino acid sequence of SEQ ID NO:10. 22.The nucleic acid sequence of claim 20, wherein the nucleic acid sequenceencodes an amino acid sequence that is more than 90% identical to theamino acid sequence of SEQ ID NO:10.
 23. The nucleic acid sequence ofclaim 20, wherein the nucleic acid sequence encodes an amino acidsequence that is more than 95% identical to the amino acid sequence ofSEQ ID NO:10.
 24. A vector comprising the nucleic acid sequence of claim20.
 25. The vector of claim 24, wherein the vector is a retroviralvector.
 26. The vector of claim 24, wherein the vector is a lentiviralvector.
 27. The vector of claim 24, wherein the vector comprises one ormore of promoters, enhancers, polyadenylation signals, transcriptionterminators, or internal ribosome entry sites (IRES).
 28. The vector ofclaim 24, wherein the vector further comprises a selectable marker gene.